Proteolysis-coupled secretion of the N terminus of apolipoprotein B. Characterization of a transient, translocation arrested intermediate.

The concept that hepatic cholesterol synthesis regulates hepatocyte assembly and secretion of apoB-containing lipoproteins remains controversial. The present study was carried out in HepG2 cells to examine the regulation of apoB secretion by the HMG-CoA reductase inhibitor atorvastatin. ApoB accumulation in the media was decreased by 24% and 36% at 10 M ( P Ͻ 0.02) and 20 M ( P Ͻ 0.01) of atorvastatin, respectively. Atorvastatin inhibited HepG2 cell cholesterol synthesis by up to 96% ( P Ͻ 0.001) and cellular cholesteryl ester (CE) mass by 54% ( P Ͻ 0.001). Another HMG-CoA reductase inhibitor, simvastatin, decreased cellular cholesterol synthesis and CE mass by up to 96% ( P Ͻ 0.001) and 52% ( P Ͻ 0.001), respectively. However, in contrast to atorvastatin, simvastatin had no effect on apoB secretion. To characterize the reduction in apoB secretion by atorvastatin (10 M ), pulse-chase experiments were performed and the kinetic data were analyzed by multicompartmental modeling using SAAM II. Atorvastatin had no affect on the synthesis of apoB, however, apoB secretion into the media was decreased by 44% ( P ‫؍‬ 0.048). Intracellular apoB degradation increased proportionately ( P ‫؍‬ 0.048). Simvastatin (10 M ) treatment did not significantly alter either the secretion or intracellular degradation of apoB, relative to control. The kinetics of apoB degradation were best described by a rapidly and a slowly turning over degradation compartment. The effect of atorvastatin on apoB degradation was largely confined to the rapid compartment. Neither inhibitor affected apoB mRNA concentrations, however, both significantly increased LDL receptor and HMG-CoA reductase mRNA levels. Atorvastatin treatment also decreased the mRNA for the microsomal triglyceride transfer protein (MTP) by 22% ( P Ͻ 0.02). These results show that atorvastatin decreases apoB secretion, by a mechanism that results in an enhanced intracellular degradation of apoB. -Wilcox, L. J., P. H. R. Barrett, and M. W. Huff. Differential regulation of apolipoprotein B secretion from HepG2 cells by two HMG-CoA reductase inhibitors, atorvastatin and simvastatin.

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confidence: 99%

Differential regulation of apolipoprotein B secretion from HepG2 cells by two HMG-CoA reductase inhibitors, atorvastatin and simvastatin

Wilcox

Barrett

Huff

1999

“…This model is based primarily on the behavior of apoB, which, under conditions of limiting lipid or MTP, undergoes misfolding and subsequent degradation by the ubiquitin/proteasome-mediated pathway (15,52). The results presented here, however, demonstrate efficient N-linked glycosylation at sites downstream of all domains reported to mediate apoB's inefficient translocation (10,27,28). As the active site for oli-gosaccharyltransferase resides in the lumen of the ER (31,53), these results suggest that the forward translocation of apoB into the ER may be an efficient and essentially unregulated process.…”

Section: Discussionmentioning

confidence: 78%

“…Unstimulated HepG2 cells degrade ϳ 75-90% of newly synthesized apoB-100 (43). This percentage is similar to the amount of apoB reported to be accessible to exogenous proteases (10,11,19,44). We sought to confirm the existence of untranslocated apoB by monitoring glycosylation site utilization at positions downstream of putative sites of translocation arrest.…”

Section: Intracellular and Secreted Forms Of Hepg2 Cell Apob-100 Are Glycosylated To The Same Extentmentioning

confidence: 81%

“…The translocation arrest model is based primarily on apoB's accessibility to exogenously added proteases and antibodies in microsomes and permeabilized cells (11, 17 -20). In HepG2 cells, approximately 75% of apoB-100 is reported to display cytosolic domains at steady state (10,11,19). However, efforts to identify the domains that specify apoB's reduced translocation efficiency have not yielded a consensus nor has a mechanism been proposed to account for how cotranslational assembly events in the ER lumen regulates apoB's translocation efficiency.…”

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confidence: 99%

See 1 more Smart Citation

Efficient glycosylation site utilization by intracellular apolipoprotein B: implications for proteasomal degradation

Huang¹,

Shelness

1999

The balance between the hepatic assembly of apolipoprotein B (apoB) and its presecretory degradation at the level of the endoplasmic reticulum (ER) may control the secretion of apoB-containing lipoproteins. In one model, apoB that fails to assemble with lipid undergoes translocation arrest, exposing the protein to the cytosolic proteasome. To examine apoB's translocation behavior under various metabolic conditions, glycosylation site utilization studies were performed. A 70-amino acid peptide containing three sites for N-linked glycosylation was appended to the C-terminus of apoB-50 (amino-terminal 50% of apoB) and expressed in both hepatic and nonhepatic cell lines. When the C-terminal reporter peptide was released by cyanogen bromide cleavage, all of the sites were glycosylated irrespective of cell type, labeling time, or assembly status. Similar peptide mapping of endogenous apoB-100 expressed in HepG2 cells was performed to monitor glycosylation at Asn residues 2752 (apoB-61), 2955 (apoB-65), and 3074 (apoB-68). N-linked glycosylation occurred at a minimum of two of the three sites, a frequency identical to that observed in apoB-100 recovered from cell media. Treatment of cells with proteasome inhibitors produced a 2.5-fold increase in intracellular apoB but failed to cause accumulation of an unglycosylated form.These results indicate that 1 ) the efficient translocation of apoB into the ER occurs independently of microsomal triglyceride transfer protein and its assembly with lipid and 2 ) despite its large size and affinity for lipid, delivery of misassembled apoB to the proteasome requires retrograde translocation from the ER lumen to cytosol. -Huang, X. F., and G. S. Shelness. Efficient glycosylation site utilization by intracellular apolipoprotein B: implications for proteasomal degradation.

“…In addition to the 116 kDa fragment of apoB, human aortic endothelial cells (HAEC) also produce an 85 kD protein that binds with lipoprotein lipase (43). A similar sized (85 kD) N-terminal fragment of apoB has also been reported to be secreted by CHO cells transfected with apoB cDNA that synthesizes apoB-53 (45). These CHO cells expressed apoB-53 intracellularly, but secreted mostly apoB-15 (85 kD fragment) into the media.…”

Section: Mutant Cells Produce An N-terminal Apob Fragment Of 85 Kd Sizementioning

confidence: 99%

Molecular bases of low production rates of apolipoprotein B-100 and truncated apoB-82 in a mutant HepG2 cell line generated by targeted modification of the apolipoprotein B gene

Srivastava¹,

Srivastava²,

Averna

et al. 1999

In subjects with familial hypobetalipoproteinemia heterozygous for truncated forms of apolipoprotein B, both apoB-100 and the truncated forms are produced at lower than expected rates. We studied the mechanism of low levels of apoB in a cell model produced by targeted modification of the apob gene of HepG2 cells. One of the three alleles of apob was found to be targeted. The targeted cells expressed apoB-100 and B-82. The media of mutant cells contained 56% of the levels of apoB-100 present in the media of wild-type (WT) HepG2 cells. ApoB-82 was present at 11% of the apoB-100 levels in mutant cell media. An 85-kD protein (apoB-15) representing the N-terminal fragment of apoB was also secreted, but only in the mutant cell media. We examined the mechanism of low levels of apoB-82. Cellular apoB-82 mRNA was 11% of apoB-100 mRNA, lower than the 33% expected, but consistent with relative levels of apoB-82 in the media. ApoB mRNA transcription in WT and the mutant cells did not differ, while the levels of apoB-82 mRNA in nuclei and polysomes were 46% and 12% of the levels of apoB-100 mRNA, respectively, suggesting that the lower levels of apoB-82 mRNA were due to altered message stability. In a pulse/chase experiment with [ 35 S] methionine, at zero time of chase, the amounts of apoB-100 in mutant cells was 66% that of WT levels, consistent with the modification of one allele. The fractions of newly synthesized apoB-100 secreted into the media at 2 h were 10% in the mutant cells and 19% in the WT cells, suggesting greater presecretory degradation of apoB-100 in the mutant cells. Thus, low levels of mutant apoB-82 mRNA gave rise to the low levels of apoB-82, while low levels of apoB-100 were due to low rates of secretion. -Srivastava, R. A. K., N. Srivastava, M. Averna, A. B. Cefalu, and G. Schonfeld. Molecular bases of low production rates of apolipoprotein B-100 and truncated apoB-82 in a mutant HepG2 cell line generated by targeted modification of the apolipoprotein B gene.